Currently, several techniques are known for performing the measurement of ion concentrations in a medium.
One of the most used techniques is the measurement with ion-selective electrodes (ISEs). More specifically, these electrodes comprise a selective membrane that generates an electric potential by exchanging the ions in the solution with this membrane. Currently, several types of selective membranes are known, such as crystalline membranes, glass membranes, or resin membranes. To perform the measurement of the electric potential, these electrodes require an internal reference electrode, that is immersed into a reference solution, and an external electrode immersed into the solution to be measured.
Another type of widely known sensors are the ion-sensitive field-effect transistor (ISFET) sensors. These transistors are based on field-effect transistors and usually comprise three terminals: one gate, one drain and one source. More specifically, these sensors are made on integrated circuitry (chip) and comprise a reference electrode that is not integrated in the chip. This sensor varies its threshold voltage according to the ion concentration of the solution where it is immersed into; specifically, it varies its voltage according to the ions in contact with its gate. This gate is made of a membrane that is selective to at least one type of ion. So, depending on the chosen membrane, the transistor will respond to a specific type of ion. The aforementioned threshold voltage is defined as the minimum voltage difference between the reference electrode and the source required to create a current flow between the source and the drain.
Both the sensors based on ISE electrodes and the sensors based on ISFET require a reference electrode, which is not integrated in the sensor, to measure ions. Hence, they are expensive to produce and need periodic maintenance.
Finally, there is an alternative way to perform the measurement of pH that requires no reference electrodes and that includes two ISFET transistors. In particular, one of the ISFET performs the ion measurement in the solution to be measured through its gate. Meanwhile, the gate of the other ISFET, which is sensitive to pH, remains exposed to a constant pH concentration with the incorporation of a structure covering the gate as a kind of micro-reservoir. This micro-reservoir is filled with a reference solution, which is a buffer solution at a specific pH level, and is connected to the outside, i.e. with the solution to be measured, by a micro-channel through which a liquid junction between the two solutions occurs. In this way, we obtain a reference ISFET transistor, commonly known as REFET.
Specifically, the aforementioned liquid junction allows a small potential difference between the solution to be measured and the reference solution, and thus the differential measurement of the ISFET and REFET depends chiefly on the response of the ISFET to pH or on the concentration of other ions in the solution to be measured.
A known example of this embodiment consists on the formation of a micro-reservoir sealed with an epoxy resin containing directly the reference solution, or containing a gel that has previously absorbed this reference solution. This micro-reservoir allows the reference solution to keep in contact with the REFET gate. Additionally, a glass capillary tube allows the contact between the reference solution and the solution to be measured.
The problem with this type of sensors is its useful life, as it depends on the volume of the micro-reservoir and the volume of the micro-channel. This is due to the fact that the reference solution in the micro-reservoir will be diluted and/or contaminated through the micro-channel, and so the error in the measurement will increase progressively as the pH level inside the micro-reservoir is no longer properly buffered and varies more strikingly during the performance of the measurement. For this reason it is considered a sensor with short useful life.
Another problem with this type of sensor is that when it is stored in a dry environment for a long period of time, the reference solution in the micro-reservoir evaporates slowly through the micro-channel and is replaced by air. Consequently, either the reference solution evaporates completely or air bubbles appear in the reference solution when the sensor is immersed again into the solution to be measured, hampering the sensor to work properly. For example, if the air bubbles remain in the surface of the REFET gate, or if they jam in the micro-channel obstructing the liquid junction between the outside and the inside of the micro-reservoir, the measurement of the sensor will be incorrect.
To solve these problems, there is another type of ISFET-REFET differential sensor which allows the renewal of the reference solution contained in the micro-reservoir. To that end, the micro-reservoir and the micro-channel of this sensor are fully filled with a gel coating the ISFET gate that makes up the REFET. This configuration prevents the formation of air bubbles in the micro-channel and in the micro-reservoir, and hence it avoids this problem of malfunctioning. Besides, this ISFET-REFET differential sensor allows being stored dry until its first use, or after several uses.
More specifically, the gel is soaked with the reference solution, into which the sensor has been previously immersed, and does the same function as if it contained the reference solution directly, but avoiding the formation of air bubbles in the REFET gate or in the micro-channel, hereinafter referred to as “REFET gate”.
That is, this ISFET-REFET sensor comprises a gel to contain the reference solution inside the micro-reservoir and the micro-channel with the aim of preventing problems with air bubbles and allowing a quick rehydration if the gel gets dry.
In particular, this type of sensor, for its correct use, must be first immersed into a reference solution so that the gel can absorb it. Once the REFET is filled with the solution, the calibration can be made in the same reference solution. After this step, measurements are performed to compare the ion concentration in the solution to be measured with the ion concentration in the reference solution. Subsequently, if needed, the sensor can be immersed again into the reference solution of the same type for calibrating again the sensor and for allowing the solution in the micro-reservoir, which may have varied slightly during the measurement, to renew by diffusion through the channel.
Despite these advantages, this sensor presents a significant error in the measurement of electric potential. This is due to the fact that additional potential arises between the REFET gate and the ISFET gate. Specifically, this potential arises between the gel and the external solution to be measured. The said potential, known as Donnan potential, which depends on the concentration of the different ions in the solution to be measured, causes an interference in the measurement, and thus reduces the sensor's accuracy.
The abovementioned interferences are evidenced in FIG. 1a and FIG. 1b, where two ISFET-REFET differential sensors have been immersed into two buffer solutions with pH 7 and pH 4. In particular, FIG. 1a shows the results obtained with a REFET the micro-reservoir and micro-channel of which are totally filled with poly(HEMA)-type gel, and which has been previously soaked with a buffer solution with pH 7. In contrast, FIG. 1b shows the results obtained with a REFET the micro-reservoir and micro-channel of which are totally filled with the buffer solution with pH 7.
To apply the gate voltage of both REFETs, a reference electrode has been used in order to ensure variations in the potential of the solution with maximum values of 1-2 mV. The applied gate voltage shown in the Figures corresponds to that maintained by a drain constant current of 100 μA and a drain-to-source constant voltage of 0.5 V. In this way, the variations in the transistor's threshold voltage are faithfully reflected in the applied gate voltage.
As shown in FIGS. 1a and 1b, the response of the REFET filled with hydrogel has high variations of potential in the order of 10 mV with significant variations in time, while in the case of the REFET without hydrogel it has lower variations of potential in the order of 2 mV and no significant variations in time.
There exists also, according to what is described in the document U.S. Pat. No. 4,874,499, a sensor with a configuration made up of a first ion sensor comprising an ion-sensitive membrane in the opening of a cavity in the first sensor, and a second sensor that acts as REFET with a porous membrane providing an opening in the cavity of the second sensor—the aim of this porous membrane is to allow a liquid junction between the inside and the outside of the cavity.